Cell line for producing retrovirus as well as preparation and use methods thereof

Abstract

The invention relates to a cell line for producing retrovirus and a preparation and use method thereof. In particular, the present invention provides a cell for producing a virus, said cell expressing RD114, Gag and Pol. The constructed cell line is good in stability, the retrovirus can be continuously and efficiently produced, and the produced virus can efficiently infect cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the construction and application of γ-retroviral toxin-producing cells. Background Technology

[0002] Gamma retroviruses are a class of viruses capable of reverse transcription and integration into the host cell genome. Under the action of reverse transcriptase, their genomic RNA can be reverse transcribed into proviral DNA, which is then inserted into the host cell genome via the LTRs at both ends of the provirus using integrase. Generally, simple retroviruses contain three main coding segments and a small coding domain. The main segments contain three genes—gag, pol, and env. gag encodes the viral core protein, pol encodes reverse transcriptase, and env encodes the viral outer membrane glycoprotein. As one of the earliest developed viral vectors, retroviral vectors have undergone numerous improvements. Currently, they only have the ability to cause a single infection; the infected host cells cannot produce replicable viruses, and the pathogenicity of the viral vectors has been greatly reduced. They are commonly used as tools for gene transfer in fields such as cell therapy. Currently, there are some known gamma retrovirus-producing cell lines, but they have some technical limitations, such as poor cell line stability, the need for inducing agents, and a relatively high amount of impurities in the virus harvesting fluid. Summary of the Invention

[0003] To overcome the above problems, this invention proposes a method for constructing and culturing γ-retroviral cell lines.

[0004] A first aspect of the present invention provides a cell for producing a virus, said cell expressing RD114, Gag and Pol and capable of assembling them into a virus.

[0005] In one or more embodiments, the cell contains nucleic acid sequences encoding RD114, Gag, and / or Pol.

[0006] In one or more embodiments, nucleic acid sequences encoding RD114, Gag, and / or Pol are integrated into the cell genome.

[0007] In one or more embodiments, the cell comprises an expression cassette containing a nucleic acid sequence encoding RD114 operatively linked to a promoter. In one or more embodiments, the amino acid sequence of RD114 is shown in SEQ ID NO:2. In one or more embodiments, the coding sequence of RD114 is operatively linked to an SFFV promoter. In one or more embodiments, the expression cassette further comprises a gag coding sequence for FeLV and a FeLV LTR, preferably located at the 5' and 3' ends of RD114, respectively. In one or more embodiments, the expression cassette is introduced into the cell by the pNT3.6_SFFV_RD114 plasmid. In one or more embodiments, the expression cassette is shown in SEQ ID NO:1.

[0008] In one or more embodiments, the cell comprises an expression cassette containing nucleic acid sequences encoding Gag and / or Pol that are operatively linked to a promoter. In one or more embodiments, the amino acid sequences of Gag-Pol are as shown in SEQ ID NO:4. In one or more embodiments, the nucleic acid sequences encoding Gag and / or Pol are integrated into the AAVS1 site of the cell genome. In one or more embodiments, the nucleic acid sequences encoding Gag and / or Pol are operatively linked to a CMV promoter. In one or more embodiments, the nucleic acid sequences encoding Gag and / or Pol are also operatively linked to a CMV enhancer. In one or more embodiments, the expression cassette is introduced into the cell by a BT-P430 (GPRV11) plasmid. In one or more embodiments, the expression cassette is as shown in SEQ ID NO:3.

[0009] In one or more embodiments, the virus is a retrovirus, preferably a gamma retrovirus.

[0010] In one or more embodiments, the viral vector is preferably selected from mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), mouse leukemia virus (MLV), and / or MSGV.

[0011] In one or more embodiments, the cells are HEK293 cells, including HEK293T, HEK293H, HEK293F, HEK293S, HEK293T / 17, HEK293T / 17SF, HEK293FT, HEK293SG, HEK293E, HEK293-6E, HEK293FTM, and HEK293SGGD cells.

[0012] In one or more embodiments, the cells are cells with accession number CCTCC NO:C202517 and / or cells with accession number CCTCC NO:C202518.

[0013] A second aspect of the invention also provides a cell for producing a virus containing a sequence of interest, said cell expressing RD114, Gag, and Pol, said sequence of interest being integrated into the cell genome.

[0014] In one or more embodiments, the sequence of interest is operationally connected to one or more elements selected from the following: 5′LTR (Long Terminal Repeat), SD (Splicing Donor), SA (Splicing Acceptor), and 3′LTR.

[0015] In one or more embodiments, the sequence of interest comprises the coding sequence of the TCR. In one or more embodiments, the alpha chain of the TCR is as shown in SEQ ID NO:6, and / or, the beta chain of the TCR is as shown in SEQ ID NO:7.

[0016] In one or more embodiments, the sequence of interest is shown as SEQ ID NO:5.

[0017] In one or more embodiments, the cell contains nucleic acid sequences encoding RD114, Gag, and / or Pol.

[0018] In one or more embodiments, nucleic acid sequences encoding RD114, Gag, and / or Pol are integrated into the cell genome.

[0019] In one or more embodiments, the cell comprises an expression cassette containing a nucleic acid sequence encoding RD114. In one or more embodiments, the amino acid sequence of RD114 is shown in SEQ ID NO:2. In one or more embodiments, the coding sequence of RD114 is operatively linked to an SFFV promoter. In one or more embodiments, the expression cassette further comprises a gag coding sequence for FeLV and a FeLV LTR, preferably located at the 5' and 3' ends of RD114, respectively. In one or more embodiments, the expression cassette is introduced into the cell by the pNT3.6_SFFV_RD114 plasmid. In one or more embodiments, the expression cassette is shown in SEQ ID NO:1.

[0020] In one or more embodiments, the cell comprises an expression cassette containing nucleic acid sequences encoding Gag and / or Pol. In one or more embodiments, the amino acid sequences of Gag-Pol are as shown in SEQ ID NO:4. In one or more embodiments, the nucleic acid sequences encoding Gag and / or Pol are integrated into the AAVS1 site of the cell genome. In one or more embodiments, the nucleic acid sequences encoding Gag and / or Pol are operatively linked to a CMV promoter. In one or more embodiments, the nucleic acid sequences encoding Gag and / or Pol are also operatively linked to a CMV enhancer. In one or more embodiments, the expression cassette is introduced into the cell by a BT-P430 (GPRV11) plasmid. In one or more embodiments, the expression cassette is as shown in SEQ ID NO:3.

[0021] In one or more embodiments, the virus is a retrovirus, preferably a gamma retrovirus.

[0022] In one or more embodiments, the cells are HEK293 cells, including HEK293T, HEK293H, HEK293F, HEK293S, HEK293T / 17, HEK293T / 17SF, HEK293FT, HEK293SG, HEK293E, HEK293-6E, HEK293FTM, and HEK293SGGD cells.

[0023] In one or more embodiments, the cell is a cell obtained by integrating the sequence of interest into the genome of a cell with accession number CCTCC NO:C202517. In one or more embodiments, the cell is a cell with accession number CCTCC NO:C202518.

[0024] The present invention provides a method for preparing the cells described in the first aspect herein, the method comprising: introducing a nucleic acid construct containing a nucleic acid sequence encoding RD114, Gag and / or Pol into the cells, said nucleic acid sequence being operatively linked to a promoter.

[0025] In one or more embodiments, the cell comprises an expression cassette containing a nucleic acid sequence encoding RD114. In one or more embodiments, the amino acid sequence of RD114 is shown in SEQ ID NO:2. In one or more embodiments, the coding sequence of RD114 is operatively linked to an SFFV promoter. In one or more embodiments, the expression cassette further comprises a gag coding sequence for FeLV and a FeLV LTR, preferably located at the 5' and 3' ends of RD114, respectively. In one or more embodiments, the expression cassette is introduced into the cell by the pNT3.6_SFFV_RD114 plasmid. In one or more embodiments, the expression cassette is shown in SEQ ID NO:1.

[0026] In one or more embodiments, the cell comprises an expression cassette containing nucleic acid sequences encoding Gag and / or Pol. In one or more embodiments, the amino acid sequences of Gag-Pol are as shown in SEQ ID NO:4. In one or more embodiments, the nucleic acid sequences encoding Gag and / or Pol are integrated into the AAVS1 site of the cell genome. In one or more embodiments, the nucleic acid sequences encoding Gag and / or Pol are operatively linked to a CMV promoter. In one or more embodiments, the nucleic acid sequences encoding Gag and / or Pol are also operatively linked to a CMV enhancer. In one or more embodiments, the expression cassette is introduced into the cell by a BT-P430 (GPRV11) plasmid. In one or more embodiments, the expression cassette is as shown in SEQ ID NO:3.

[0027] In one or more embodiments, the virus is a retrovirus, preferably a gamma retrovirus.

[0028] In one or more embodiments, the cells are HEK293 cells, including HEK293T, HEK293H, HEK293F, HEK293S, HEK293T / 17, HEK293T / 17SF, HEK293FT, HEK293SG, HEK293E, HEK293-6E, HEK293FTM, and HEK293SGGD cells.

[0029] In one or more embodiments, the cells are cells of CCTCC NO:C202518.

[0030] This invention provides a method for preparing the cells described in the second aspect herein, the method comprising:

[0031] (1) Introducing a nucleic acid construct containing nucleic acid sequences encoding RD114, Gag, and / or Pol into cells, wherein the nucleic acid sequences are operatively linked to a promoter.

[0032] (2) The sequence of interest is introduced into the cell obtained by (1), preferably into the cell genome.

[0033] (3) Screening cells, the cells being:

[0034] (a) The genome contains sequences of interest.

[0035] (b) It can express proteins encoded by sequences of interest.

[0036] (c) The virus produced by the cell contains a sequence of interest, or

[0037] (d) After a virus produced by the cell infects another cell, the other cell is able to express a protein encoded by a sequence of interest.

[0038] In one or more embodiments, step (2) includes the step of introducing the sequence of interest into the cell genome using any of the following technologies: retrovirus, transposon, zinc finger nuclease (ZFN) technology, transcription activator effector nuclease (TALEN) technology, and CRISPR / Cas system.

[0039] In one or more embodiments, step (2) includes infecting the cells obtained by (1) with a retrovirus containing the sequence of interest.

[0040] In one or more embodiments, the retrovirus described in step (2) comprises Gag protein, Pol protein and RD114 protein.

[0041] In one or more embodiments, the nucleic acid construct comprising the nucleic acid sequence encoding RD114 is an expression cassette. In one or more embodiments, the amino acid sequence of RD114 is shown in SEQ ID NO:2. In one or more embodiments, the coding sequence of RD114 is operatively linked to the SFFV promoter. In one or more embodiments, the expression cassette further comprises a gag coding sequence for FeLV and a FeLV LTR, preferably located at the 5' and 3' ends of RD114, respectively. In one or more embodiments, the expression cassette is introduced into cells using the pNT3.6_SFFV_RD114 plasmid. In one or more embodiments, the expression cassette is shown in SEQ ID NO:1.

[0042] In one or more embodiments, the nucleic acid construct comprising nucleic acid sequences encoding Gag and / or Pol is an expression cassette. In one or more embodiments, the amino acid sequence of Gag-Pol is as shown in SEQ ID NO:4. In one or more embodiments, the nucleic acid sequence encoding Gag and / or Pol is integrated into the AAVS1 site of the cell genome. In one or more embodiments, the nucleic acid sequence encoding Gag and / or Pol is operatively linked to a CMV promoter. In one or more embodiments, the nucleic acid sequence encoding Gag and / or Pol is also operatively linked to a CMV enhancer. In one or more embodiments, the expression cassette is introduced into cells using the BT-P430(GPRV11) plasmid. In one or more embodiments, the expression cassette is as shown in SEQ ID NO:3.

[0043] In one or more embodiments, the sequence of interest is operationally connected to one or more elements selected from the following: 5′LTR (Long Terminal Repeat), SD (Splicing Donor), SA (Splicing Acceptor), and 3′LTR.

[0044] In one or more embodiments, the sequence of interest comprises the coding sequence of the TCR. In one or more embodiments, the alpha chain of the TCR is as shown in SEQ ID NO:6, and / or, the beta chain of the TCR is as shown in SEQ ID NO:7.

[0045] In one or more embodiments, the sequence of interest is shown as SEQ ID NO:5.

[0046] In one or more embodiments, the method includes the following steps:

[0047] (1) Constructing HEK293-RD114 cells;

[0048] (2) Constructing HEK293-RD114-GP cells;

[0049] (3) Construct the original toxin-producing cell line HEK293-RD114-GP-P037 TCR.

[0050] In one or more embodiments, (1) includes the steps of: co-transfecting cells with pNT3.6_SFFV_RD114 plasmid and BT-P139 (pMAX-SB100) plasmid, and integrating RD114 into the cell genome using the Sleeping Beauty transposon system.

[0051] In one or more embodiments, step (2) includes the steps of: co-transfecting the HEK293-RD114 cells with the BT-P283 (pX330-sgAAVS1) plasmid and the BT-P430 (GPRV11) plasmid, and inserting the Gag-Pol gene into the HEK293-RD114 cells. Preferably, the insertion method includes using a CRISPR-Cas9 gene editing system.

[0052] In one or more embodiments, step (3) includes the following steps:

[0053] (i) HEK293 cells were co-transfected with BT-P037 recombinant plasmid, Gag-Pol plasmid and VSV-G plasmid to obtain retroviral solution;

[0054] (ii) The HEK293-RD114-GP cells were infected with retroviral fluid, and the target gene sequence in the BT-P037 recombinant plasmid was integrated into the genome of the HEK293-RD114-GP cells.

[0055] In one or more embodiments, the method further includes step (4): acclimatizing and culturing the toxin-producing cell line of (3).

[0056] In one or more embodiments, the suspension acclimatization culture is a serum-free suspension acclimatization culture.

[0057] The present invention also provides a method for preparing or rescuing a virus, comprising preparing the virus using cells described in any embodiment of the first aspect herein.

[0058] In one or more embodiments, the virus is a retrovirus, preferably a gamma retrovirus.

[0059] In one or more embodiments, the cells are HEK293 cells, including HEK293T, HEK293H, HEK293F, HEK293S, HEK293T / 17, HEK293T / 17SF, HEK293FT, HEK293SG, HEK293E, HEK293-6E, HEK293FTM, and HEK293SGGD cells.

[0060] In one or more embodiments, the method includes:

[0061] (1) The sequence of interest is introduced into the cells described in the first aspect of this document (e.g., cells with accession number CCTCCNO:C202517), preferably integrated into the cell genome, and

[0062] (2) Incubate the cells under conditions suitable for virus production.

[0063] In one or more embodiments, step (1) includes the step of introducing the sequence of interest into the cell genome using any of the following technologies: retrovirus, transposon, zinc finger nuclease (ZFN) technology, transcription activation effector nuclease (TALEN) technology, and CRISPR / Cas system.

[0064] In one or more embodiments, step (1) includes infecting the cells with a retrovirus containing a sequence of interest.

[0065] The present invention also provides the use of the cells described in any embodiment of the present invention in the preparation of viruses, increasing virus titers, and improving virus packaging efficiency.

[0066] In one or more embodiments, the virus is a retrovirus, preferably a gamma retrovirus.

[0067] In one or more embodiments, the cells are HEK293 cells, including HEK293T, HEK293H, HEK293F, HEK293S, HEK293T / 17, HEK293T / 17SF, HEK293FT, HEK293SG, HEK293E, HEK293-6E, HEK293FTM, and HEK293SGGD cells.

[0068] The present invention also provides a kit comprising the cells described in the first aspect herein.

[0069] The present invention also provides a pharmaceutical composition comprising a gamma retrovirus prepared by the method described in any embodiment herein and pharmaceutically acceptable excipients.

[0070] The present invention also provides the use of cells described in any embodiment of the invention or viruses prepared by the methods described in any embodiment of the invention in the preparation of pharmaceuticals.

[0071] In one or more embodiments, the virus is a retrovirus, preferably a gamma retrovirus.

[0072] In one or more embodiments, the drug is an immunotherapy drug.

[0073] In one or more embodiments, the drug comprises cells transfected by the virus, preferably immune cells.

[0074] In one or more embodiments, the drug contains the virus.

[0075] In one or more embodiments, the immune cells contain a sequence of interest, preferably a coding sequence for a CAR or TCR.

[0076] In one or more embodiments, the immune cells are T cells or NK cells.

[0077] Advantages of this invention:

[0078] 1. The constructed cell line exhibits good stability and can sustainably and efficiently produce gamma retroviruses;

[0079] 2. The produced gamma retrovirus is a virus with the RD114 envelope protein, which can efficiently infect human cells such as T cells;

[0080] 3. It has broad application prospects in fields such as gene therapy and cell therapy. Attached Figure Description

[0081] Figure 1 Image of the H-P16 (pNT3.6_SFFV_RD114) plasmid.

[0082] Figure 2 BT-P139(pMAX-SB100) plasmid map.

[0083] Figure 3 Detection of RD114 expression in HEK293-RD114 mixed clone cells.

[0084] Figure 4 Detection of RD114 expression in HEK293-RD114 monoclonal cell line.

[0085] Figure 5 Results of viral packaging ability of HEK293-RD114 monoclonal cell line.

[0086] Figure 6 Results of viral packaging ability of HEK293-RD114 monoclonal cell line.

[0087] Figure 7 Image of BT-P283(pX330-sgAAVS1) plasmid.

[0088] Figure 8 BT-P430(GPRV11) plasmid map.

[0089] Figure 9 : Direct PCR identification results of HEK293-RD114-GP monoclonal cell line.

[0090] Figure 10 PCR identification results of genomic DNA from HEK293-RD114-GP monoclonal cell line.

[0091] Figure 11Gag-Pol plasmid map.

[0092] Figure 12 VSV-G plasmid map.

[0093] Figure 13 Image of the BT-P037 recombinant plasmid.

[0094] Figure 14 Detection of expression of P037 TCR in the original toxin-producing strain HEK293-RD114-GP-P037 TCR.

[0095] Figure 15 Detection of P037 TCR expression in HEK293-RD114-GP-P037 TCR monoclonal cell line.

[0096] Figure 16 Detection of toxin production capacity of HEK293-RD114-GP-P037 TCR monoclonal cell line.

[0097] Figure 17 Comparison of toxin production capacity of HEK293-RD114-GP-P037 TCR monoclonal cell lines.

[0098] Figure 18 : PBMC validation of the toxin-producing ability of the HEK293-RD114-GP-P037 TCR monoclonal cell line.

[0099] Figure 19 Optimization of harvest time for suspension culture of HEK293-RD114-GP-P037 TCR toxin-producing cell line.

[0100] Figure 20 Results of cell viability assays at different passages of HEK293-P037clone46 cells

[0101] Figure 21 Results of viable cell density assay at different passages of HEK293-P037clone46 cells

[0102] Figure 22 Results of target gene copy number detection at different passages in HEK293-P037clone46 cells

[0103] Figure 23 Results of virus particle count detection at different passages in HEK293-P037clone46 cells

[0104] Figure 24 Results of viral transduction titer assay in HEK293-P037clone46 cells at different passages

[0105] Figure 25Results of viral activity assays at different passages in HEK293-P037clone46 cells Detailed Implementation

[0106] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0107] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0108] One objective of this invention is to provide a method for constructing and culturing a gamma retrovirus-producing cell line. First, the RD114 gene is integrated into the HEK293 cell genome using the Sleeping Beauty transposon system. Then, the Gag-Pol gene is integrated into the HEK293-RD114 cell genome using CRISPR-Cas9 gene editing. Finally, the target gene is integrated into the HEK293-RD114-GP cell genome using a VSV-G envelope protein retrovirus. This constructs a stable gamma retrovirus-producing cell line. After serum-free suspension culture, serum-free suspension culture of the gamma retrovirus-producing cell line is achieved. The culture supernatant is harvested and purified efficiently using high-speed centrifugation or column chromatography. The purified gamma retrovirus can be used as a raw material for cell genetic modification.

[0109] A first aspect of the present invention provides a cell for producing viruses, said cell expressing RD114, Gag, and Pol and capable of assembling them into viruses. The cell contains nucleic acid sequences encoding RD114, Gag, and / or Pol, or these nucleic acid sequences are integrated into the cell genome. The present invention also provides cells for producing viruses containing sequences of interest, said cells expressing RD114, Gag, and Pol, said sequences of interest being integrated into the cell genome.

[0110] To express these coding sequences, they typically need to be operationally linked to a promoter. The coding sequence can be expressed using a promoter in the cell's genome, or it can be operationally linked to a promoter to form an insert sequence. In this paper, the promoter operationally linked to the protein's coding sequence can be any promoter suitable for initiating protein expression in cells (e.g., HEK293 cells), and these promoters are conventional techniques in the art, such as the CMV promoter, SFFV promoter, CAG promoter, etc.

[0111] In this paper, coding sequences or contained insert sequences can be integrated into any location in the genome, as long as they can be expressed in cells and do not affect normal cell growth and function. This can be achieved through various methods, such as integrating coding sequences into specific locations in the genome, or randomly integrating coding sequences into the genome and screening cells. Methods for integrating insert sequences into specific locations in the genome are known in the art, such as using gene editing techniques based on CRISPR / Cas enzymes to insert the sequence. Methods for randomly integrating coding sequences into the genome and screening cells are also known in the art, such as using homologous recombination, transposon systems, etc. Specifically, methods for introducing sequences of interest into the cell genome include, but are not limited to: retroviruses, transposons, zinc finger nuclease technology (ZFN), transcription activator effector nuclease (TALEN) technology, and CRISPR / Cas systems.

[0112] Methods for introducing insert sequences into cells mainly include plasmid transformation or direct introduction of mRNA. Plasmids are a common technique in this field.

[0113] Vectors can be constructed using techniques conventional in the art. A typical expression vector contains expression control sequences that can be used to regulate the expression of the desired nucleic acid sequence. In recombinant expression vectors, "operational ligation" refers to the connection of the target nucleotide sequence to a regulatory sequence in a manner that allows the nucleotide sequence to be expressed. Those skilled in the art are familiar with methods for constructing expression vectors containing the coding sequence of the fusion protein of this invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. Vectors suitable for use herein can be bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. Any plasmid and vector can be used as long as it can replicate and remain stable in the host (especially HEK293). An important characteristic of expression vectors is that they typically contain regulatory sequences, including but not limited to origin of replication, promoters, enhancers, marker genes, translation control elements, ribosome binding sites for translation initiation, and transcription terminators. Representative promoters include: the lac or trp promoter of Escherichia coli; the PL promoter of λ phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the LTRs of retroviruses, and some other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses.

[0114] In expression vectors, various nucleic acids and regulatory sequences can be linked together to produce recombinant expression vectors that may include one or more convenient restriction sites that allow the insertion or substitution of nucleotide sequences encoding a polypeptide at such sites. In preparing the expression vectors of the present invention, the coding sequences of various different proteins described herein are located in the vector such that the coding sequences are operatively linked for the expression of appropriate regulatory sequences.

[0115] In addition, a single vector or plasmid, or two or more vectors or plasmids, or transposons, containing the total DNA to be introduced into the host cell genome can be used. The expression vector preferably contains one or more selectable markers that allow for easy selection of cells for transformation, transfection, transduction, etc. Selectable markers are genes whose products provide resistance to antibiotics or viruses, resistance to heavy metals, prototrophic to auxotrophic traits, etc. Preferably, the expression vector contains elements that allow the vector to integrate into the host cell genome or to replicate autonomously in the cell independently of the genome.

[0116] One or more copies of the coding sequence of the present invention can be inserted into a host cell to increase the yield of the gene product. The increase in polynucleotide copy number can be achieved by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selector gene and the polynucleotide. Cells containing an amplified copy of the selector gene and thereby containing additional copies of the polynucleotide can be screened by culturing the cells in the presence of a suitable selector.

[0117] In one embodiment, the cell includes an insert sequence as an expression cassette, the expression cassette including a nucleic acid sequence encoding RD114 operatively linked to a promoter, and a nucleic acid sequence encoding Gag and / or Pol operatively linked to a promoter. The expression cassette may be one, two, or more, each containing the coding sequences for RD114, Gag, and Pol, respectively.

[0118] The expression box described herein may also include the gag encoding sequence of FeLV and the FeLV LTR, preferably located at the 5' end and 3' end of the encoding sequence, respectively.

[0119] In this document, the amino acid sequence of RD114 is shown in SEQ ID NO:2. In one or more embodiments, the coding sequence of RD114 is operatively linked to the SFFV promoter. The expression cassette containing the coding sequence of RD114 is introduced into cells by the pNT3.6_SFFV_RD114 plasmid. In one or more embodiments, the expression cassette is shown in SEQ ID NO:1.

[0120] In this document, the amino acid sequence of Gag-Pol is shown in SEQ ID NO:4. In one or more embodiments, the nucleic acid sequence encoding Gag and / or Pol is integrated into the AAVS1 site of the cell genome. In one or more embodiments, the nucleic acid sequence encoding Gag and / or Pol is operatively linked to the CMV promoter. In one or more embodiments, the expression cassette is introduced into cells by the BT-P430 (GPRV11) plasmid. In one or more embodiments, the expression cassette is shown in SEQ ID NO:3.

[0121] In this article, "retrovirus" refers to RNA viruses, which require reverse transcriptase to first convert RNA into cDNA. The newly synthesized cDNA then inserts into the host's nuclear DNA and replicates, transcribes, and translates along with the host DNA to achieve amplification. Retroviruses have three structural protein genes: the gag gene, which encodes structural proteins such as the viral capsid and matrix; the pol gene, which encodes reverse transcriptase (p66 / p51), proteolytic enzymes, and integrase; and the env gene, which encodes two envelope glycoproteins, gp120 and gp41.

[0122] For example, in this application, the pNT3.6_SFFV_RD114 plasmid contains the RD114 gene, the BT-P430(GPRV11) plasmid contains the Gag-Pol gene, and the BT-P037 plasmid contains the TCR gene targeting the KRAS G12V mutation. In this document, the stable cell lines express the RD114 gene, the Gag-Pol gene, and the TCR gene. The coding sequences described in this invention can be obtained using PCR amplification, recombination, or synthetic methods known in the art. For PCR amplification, the primer sequences and templates disclosed herein can be used. After obtaining the recombinant expression vector, the vector is transformed into host cells to produce a protein or peptide including a fusion protein. This transfer process can be performed using conventional techniques well known to those skilled in the art, such as transformation or transfection.

[0123] The host cell described in this invention can be any cell of interest, especially cells conventionally used in the art for transfecting plasmids to express exogenous genes, and can be eukaryotic cells (such as animal and plant cells) and prokaryotic cells (such as Escherichia coli and other bacteria). For example, the cell can be a human cell. Examples of cells include, but are not limited to, HEK293 cells, MDCK cells, and HeLa cells. In one or more embodiments, the cell is a HEK293 cell, including HEK293T, HEK293H, HEK293F, HEK293S, HEK293T / 17, HEK293T / 17SF, HEK293FT, HEK293SG, HEK293E, HEK293-6E, HEK293FTM, and HEK293SGGD cells.

[0124] The term "Sleeping Beauty transposon system" in this paper refers to the Sleeping Beauty (SB) transposon system, which consists of the Sleeping Beauty (SB) transposase and transposons. The SB transposon system can insert specific DNA sequences into the genome of vertebrates. DNA transposons transfer one DNA fragment to another in a cut / paste manner. Transposition is a precise process in which a specific DNA fragment is cut from a donor DNA molecule and then moved to another site in the same or different DNA molecule or genome. In this paper, the Sleeping Beauty transposon system was used to integrate RD114 into the genome of HEK293 cells.

[0125] The present invention provides a method for preparing cells that can be used to produce viruses, the method comprising: introducing a nucleic acid construct containing a nucleic acid sequence encoding RD114, Gag and / or Pol into cells, said nucleic acid sequence being operatively linked to a promoter.

[0126] The present invention provides a method for preparing cells capable of producing viruses containing a sequence of interest, the method comprising: (1) introducing a nucleic acid construct containing a nucleic acid sequence encoding RD114, Gag, and / or Pol into a cell, the nucleic acid sequence being operatively linked to a promoter; (2) introducing the sequence of interest into the cell obtained in (1), preferably into the cell genome; and (3) screening cells that: (a) have the sequence of interest integrated into their genome; (b) express a protein encoded by the sequence of interest; (c) have a virus produced by the cell containing the sequence of interest; or (d) have another cell infected by a virus produced by the cell, the other cell expressing a protein encoded by the sequence of interest.

[0127] In one or more embodiments, the method includes the following steps:

[0128] (1) Construct HEK293-RD114 cells; preferably, co-transfect cells with pNT3.6_SFFV_RD114 plasmid and BT-P139(pMAX-SB100) plasmid, and integrate RD114 into the cell genome using the Sleeping Beauty transposon system.

[0129] (2) Constructing HEK293-RD114-GP cells; preferably, co-transfecting the HEK293-RD114 cells with the BT-P283 (pX330-sgAAVS1) plasmid and the BT-P430 (GPRV11) plasmid to insert the Gag-Pol gene into the HEK293-RD114 cells. More preferably, the insertion method includes using a CRISPR-Cas9 gene editing system.

[0130] (3) Constructing the original toxin-producing cell line HEK293-RD114-GP-P037 TCR. Preferably, the process includes the following steps: (i) co-transfecting HEK293 cells with the BT-P037 recombinant plasmid, Gag-Pol plasmid, and VSV-G plasmid to obtain retroviral solution; (ii) infecting the HEK293-RD114-GP cells with the retroviral solution, so that the target gene sequence in the BT-P037 recombinant plasmid can be integrated into the genome of the HEK293-RD114-GP cells.

[0131] This invention provides a method for preparing or rescuing gamma retroviruses, comprising incubating cells as described in any embodiment herein to prepare the virus. Specifically, the method includes constructing a toxin-producing cell line that stably produces gamma retroviruses, said toxin-producing cell line being a stable cell line as described in the first aspect of this invention. In one or more embodiments, the method includes the following steps:

[0132] (1) Construct HEK293-RD114 cells; preferably, co-transfect cells with pNT3.6_SFFV_RD114 plasmid and BT-P139(pMAX-SB100) plasmid, and integrate RD114 into the cell genome using the Sleeping Beauty transposon system; (2) Construct HEK293-RD114-GP cells; preferably, co-transfect HEK293-RD114 cells with BT-P283(pX330-sgAAVS1) plasmid and BT-P430(GPRV11) plasmid, and insert the Gag-Pol gene into HEK293-RD114 cells. More preferably, the insertion method includes using a CRISPR-Cas9 gene editing system; (3) Construct the HEK293-RD114-GP-P037 TCR original toxin-producing cell line. Preferably, the method includes the following steps: (i) co-transfecting HEK293 cells with the BT-P037 recombinant plasmid, Gag-Pol plasmid, and VSV-G plasmid to obtain a retroviral solution; (ii) infecting the HEK293-RD114-GP cells with the retroviral solution, wherein the target gene sequence in the BT-P037 recombinant plasmid can be integrated into the genome of the HEK293-RD114-GP cells. In one or more embodiments, the method further includes step (4): subjecting the toxin-producing cell line from (3) to suspension acclimatization culture. Preferably, the suspension acclimatization culture is a serum-free suspension acclimatization culture.

[0133] In an implementation involving a sequence of interest, a method for preparing or rescuing a gamma retrovirus includes: (1) introducing the sequence of interest into the cell, preferably integrating it into the cell genome, and (2) incubating the cell under conditions suitable for virus production to obtain a virus containing the sequence of interest.

[0134] The present invention also provides the use of the cells described in any embodiment of the present invention in the preparation of viruses, increasing virus titers, and improving virus packaging efficiency.

[0135] The present invention also provides a kit comprising the stable cell line described in the first aspect herein.

[0136] This invention also provides a pharmaceutical composition comprising a therapeutically effective amount of gamma retrovirus prepared by the method described in any embodiment of this invention, and pharmaceutically acceptable excipients. The therapeutically effective amount is an amount that effectively treats or alleviates one or more symptoms of a disease and can be determined by those skilled in the art using conventional methods. The pharmaceutical composition may also contain a suitable pharmaceutically acceptable carrier or excipient. For example, for a pharmaceutical composition containing cells, it may contain a suitable carrier or excipient suitable for maintaining cell viability and compatibility with animal bodies. The excipients are preferably non-toxic to the recipient at the dose and concentration used. This invention also provides the use of the stable cell lines described in the first aspect of this invention in the preparation of reagents for the production of gamma retroviruses. This invention also provides the use of gamma retroviruses prepared by the method described in any embodiment of this invention in infecting human cells such as T cells, or for cell genetic modification. In one or more embodiments, the cell genetic modification includes CAR-T cell therapy, CAR-NK cell therapy, TCR-T cell therapy, etc.

[0137] The embodiments of the present invention will now be described in detail with reference to examples. Those skilled in the art will understand that the following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0138] Example

[0139] Example 1: Construction of HEK293-RD114 cells

[0140] First, HEK293 cells were co-transfected with the H-P16 (pNT3.6_SFFV_RD114) plasmid (containing the RD114 gene) and the BT-P139 (pMAX-SB100) plasmid. The sequence structure is as follows. Figure 1 and Figure 2 As shown, the RD114 insertion sequence was integrated into the genome of HEK293 cells using the Sleeping Beauty transposon system. The transposon consists of a set of mirror-image inverted repeat sequences (IR / DR), located on either side of RD114 in the plasmid backbone, and RD114 expression is driven by the SFFV promoter. The RD114 insertion sequence is shown in SEQ ID NO:1, and the RD114 protein sequence is shown in SEQ ID NO:2. RD114 expression was detected by flow cytometry. HEK293-RD114 mixed clones in good culture condition were used to establish single-clone cell lines using flow cytometry sorting. The results showed that the positive rate of RD114 expression in HEK293-RD114 mixed clones was 17.8%. (See attached table). Figure 3 .

[0141] Two weeks later, a total of 83 monoclonal cell lines were selected, and RD114 expression was detected by flow cytometry. The results are shown below. Figure 4Among them, clones 4, 5, 11, 20, 25, 28, 31, 32, 35, 36, 41, 43, 44, 51, 55, 63, 65, 71, 78, 83, and 85 showed good growth status and high and uniform RD114 expression levels.

[0142] Based on cell growth, clones 5, 20, 25, 28, 31, 35, 51, and 55 were selected for plate formation to further compare retroviral packaging capabilities. The above eight HEK293-RD114 monoclonal cells were co-transfected with a retroviral plasmid carrying green fluorescent protein (GFP) and the Gag-Pol plasmid. After 72 hours, the viral load was collected and used to infect HEK-293T reporter cells. The GFP positivity rate in reporter cells was used to represent retroviral packaging capability. The results showed significant differences in retroviral packaging capabilities among the eight clones. Clone 28 exhibited the strongest GFP positivity rate (68.7%), followed by clones 5 and 31. (See attached table). Figure 5 .

[0143] Clones 5, 28, and 31 were compared again with four other single clones (clones 32, 44, 63, and 83) using the same method. The results showed that clone 28 still exhibited the strongest retroviral packaging ability among the seven clones, with a GFP positivity rate of 33.2%. Flow cytometry results are shown below. Figure 6 .

[0144] Combining the results of the two studies, clone 28 retrovirus showed the strongest packaging ability. HEK293-RD114 cells were successfully constructed and named HEK293-RD114 clone28 cells. Further expansion culture was carried out for subsequent construction of HEK293-RD114-GP cells.

[0145] Example 2: Construction of HEK293-RD114-GP cells

[0146] HEK293-RD114 cells were co-transfected with the BT-P283 (pX330-sgAAVS1) plasmid and the BT-P430 (GPRV11) plasmid (which contains the Gag-Pol gene). The sequence structure is as follows: Figure 7 and Figure 8 As shown, the AAVS1 site on chromosome 19 was specifically cut using the CRISPR-Cas9 gene editing system, generating a DNA double-strand break. Subsequently, through homologous recombination repair, the Gag-Pol insert sequence was integrated into the AAVS1 site on the genome using the BT-P430 (GPRV11) plasmid as the donor DNA sequence. The sequence structure is shown in the figure. Figure 8As shown, the sequence is as follows: left homologous arm (HA-L), puromycin resistance gene, CMV enhancer, CMV promoter, Gag-Pol gene, and right homologous arm (HA-R). The Gag-Pol insertion sequence is shown in SEQ ID NO:3, and the Gag-Pol coding sequence is shown in SEQ ID NO:4. Cells with the inserted Gag-Pol gene were selected using puromycin on the BT-P430(GPRV11) plasmid.

[0147] Primers were designed (see Table 1) to identify whether the Gag-Pol gene was correctly integrated using PCR. Primers prim951 + prim952 amplified a 1702 bp fragment from the AAVS1 integration site upstream of HA-L to the BT-P430(GPRV11) plasmid, and primers prim986 + prim954 amplified a 2510 bp fragment from the Gag-Pol sequence on the BT-P430(GPRV11) plasmid to the AAVS1 integration site downstream of HA-R. Only cells with correct integration could amplify the target band.

[0148] Table 1. Primer information for Gag-Pol gene integration identification.

[0149]

[0150] HEK293-RD114-GP hybrid clonal cells (HEK293-RD114-GP11 cells) were identified as positive for Gag-Pol gene integration, and then monoclonal cell lines were established using a limiting dilution method. After approximately 10 days of culture, 16 monoclonal cells were selected in two batches and identified by PCR using primers prim951+prim952. Clones 4 and 8 from the first selection and clones 1 and 3 from the second selection amplified the target bands. The results are shown in the figure. Figure 9 .

[0151] These four clones were selected for expanded culture, genomic DNA was extracted, and PCR verification was performed again. All four clones were able to amplify the target bands using two sets of primers (prim951+prim952, prim986+prim964), as shown in the following figures. Figure 10 This indicates that the Gag-Pol gene has been inserted at the AAVS1 site in all four clones, and the HEK293-RD114-GP cell line was successfully constructed. Clone 8 was selected as the HEK293-RD114-GP monoclonal cell line, which is deposited at the China Center for Type Culture Collection (CCTCCNO: C202517) on December 24, 2024 (address: No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province, China).

[0152] Example 3: Construction of the original toxin-producing cell line HEK293-RD114-GP-P037 TCR

[0153] HEK293 cells were co-transfected with the BT-P037 recombinant plasmid (containing the TCR gene targeting the KRAS G12V mutation), the Gag-Pol plasmid, and the VSV-G plasmid. The sequence structure is as follows: Figure 11-13 As shown, P037 TCR VSV-G retroviral solution was obtained. HEK293-RD114-GP cells were infected with the P037 TCR VSV-G retroviral solution. The target gene sequence in the BT-P037 recombinant plasmid was integrated into the HEK293-RD114-GP cell genome. The TCR-containing insertion sequence is shown in SEQ ID NO:5, and the P037 TCR α and β chains are shown in SEQ ID NO:6-7, constructing the HEK293-RD114-GP-P037 TCR original virus-producing strain, which can continuously and stably produce retroviruses.

[0154] The expression of the original toxin-producing strain P037 TCR was detected by flow cytometry staining with anti-mTCR antibody. The results showed that the mTCR positivity rate of the prepared cells was 91.3%, indicating successful expression of P037 TCR. The original toxin-producing strain HEK293-RD114-GP-P037 TCR was successfully constructed. (See details below.) Figure 14 .

[0155] HEK293-RD114-GP-P037 TCR original toxin-producing cell line in good culture condition was used to establish monoclonal cell lines using the limiting dilution method. The growth of monoclonal cell clusters was observed under a microscope. After 13 days of culture, monoclonal cells in good condition were selected and transferred to 24-well plates for culture, resulting in a total of 65 monoclonal cell lines, which were numbered 1#-65#.

[0156] Fifty-two cell lines in good condition were selected, and P037 TCR expression was detected by flow cytometry staining with anti-mTCR antibody. The results showed that most clones were mTCR positive, with a single peak shape. Figure 15 As shown.

[0157] Cells were further seeded into new 24-well plates, 1 ml / well. Three days later, the viral fluid from the monoclonal cell line was collected to infect Jurkat reporter cells. P037 TCR expression was detected by flow cytometry staining with anti-mTCR antibody. The toxin production capacity of the HEK293-RD114-GP-P037 TCR monoclonal cell line was expressed as the mTCR positivity rate (mTCR%) in Jurkat reporter cells. Results showed that clones 16, 42, 46, and 57 exhibited the best toxin production capacity, with an mTCR positivity rate greater than 2%. (See details below.) Figure 16 .

[0158] Based on the expression level and toxin production capacity of the HEK293-RD114-GP-P037 TCR monoclonal cell line, eight monoclonal cell lines (clones 16, 26, 42, 46, 53, 56, 57, and 63) were selected and seeded into T25 cell culture flasks. Viral fluid from these monoclonal cell lines was collected twice and used to infect Jurkat reporter cells. P037 TCR expression was detected by flow cytometry staining with anti-mTCR antibody. The toxin production capacity of the HEK293-RD114-GP-P037 TCR monoclonal cell line was expressed as the mTCR positivity rate (mTCR%) in Jurkat reporter cells. Results showed that clones 42 and 46 had the highest toxin production capacity; clones 16, 26, 56, and 57 had the next highest capacity; and clones 53 and 63 had the lowest capacity. (See details below.) Figure 17 .

[0159] In addition, the toxin-producing ability of the HEK293-RD114-GP-P037 TCR monoclonal cell line was verified using human peripheral blood mononuclear cells (PBMCs). PBMCs activated with anti-CD3 antibody were infected with the monoclonal cell line virus solution. P037 TCR expression was detected by flow cytometry staining with anti-mTCR antibody. The toxin-producing ability of the HEK293-RD114-GP-P037 TCR monoclonal cell line was expressed as the mTCR positivity rate (mTCR%) in PBMC cells. The results showed that clones 16, 26, 42, 46, 56, and 57 had high toxin-producing abilities, while clones 53 and 63 had poor toxin-producing abilities. (See details below.) Figure 18 .

[0160] Based on the combined effects of the P037 TCR expression level and toxin production capacity of the HEK293-RD114-GP-P037 TCR monoclonal cell line, clone 46 showed a high positive rate of TCR expression, a single peak shape, and high viral infectivity against Jurkat reporter cells and PBMCs. Therefore, clone 46 was selected as the toxin-producing cell line (HEK293-RD114-GP-P037-clone46, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C202518 on December 24, 2024, at No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, and classified as HEK293-RD114-GP-P037) for subsequent suspension acclimatization and the establishment of a tertiary cell bank.

[0161] Example 4: Suspension acclimatization culture of HEK293-RD114-GP-P037 TCR virus-producing cell line and optimization of virus harvesting time

[0162] HEK293-RD114-GP-P037 TCR toxin-producing cells in the logarithmic growth phase were collected. After trypsin digestion at 37°C for 3 minutes, the cells were resuspended in complete culture medium. The cell suspension was centrifuged at 500g for 5 minutes. The supernatant was discarded. The cells were resuspended in OPM-293CD05 Medium to a concentration of 1×10⁻⁵. 6 Cells / ml. Transfer cells to 250ml cell culture flasks and incubate at 37°C with a shaker. Cell suspension acclimation is complete after three passages. The acclimated cell suspension is then diluted with water at 1×10⁻⁶ cells / ml. 6 Cells / ml were seeded at a density of [number] cells / ml in 250ml shake flasks. Cell culture supernatant was harvested every 24 hours for three consecutive days. The titer of gamma retrovirus in the culture supernatant was measured; see [link to details]. Figure 19 The results showed that the highest titer was found in the supernatant after 48 hours of incubation.

[0163] Example 5: Validation of passage stability of HEK293-RD114-GP-P037 TCR monoclonal toxin-producing cell line

[0164] The passage stability of the monoclonal toxin-producing cell line was tested. One cell line was taken from the working cell bank. The cells were revived in a 37°C water bath. The cell suspension was transferred to a centrifuge tube containing OPM-293CD05 Medium and centrifuged at 500g for 5 minutes. The supernatant was discarded, and the cells were resuspended in OPM-293CD05 Medium to a final volume of 3 × 10⁻⁶. 5Cells were cultured at a density of at least [number] cells / ml, and then transferred to a 250ml Erlenmeyer flask. The flask was placed in a magnetically driven stacked thermostatic shaker for culture. Culture conditions were: temperature 36-38℃, CO2 concentration 4-6%, and rotation speed 70-130 rpm. After cell resuscitation for 72±2 hours, the medium was changed. Cells were cultured for another 48±2 hours before passage. Thereafter, medium changes and passages were performed alternately every 48±2 hours. After resuscitation, the cell passage number was increased by one from the frozen passage number, designated as P13. Each subsequent passage increased the cell passage number by one. During cell culture, once cells were successfully passaged to P19, they underwent a 48±2 hour culture period. After this initial culture phase, the first medium change was performed to ensure a suitable cell growth environment. Following this, the culture conditions were maintained for another 48±2 hours without any additional operations. Sampling was then performed, and the cell medium was changed again. The cell passage represented by this sampling was clearly labeled P19. Subsequently, the cells continued to be cultured under the same conditions for another 48±2 hour cycle, followed by final sampling, at which point the cell culture process for this batch was terminated. This sampled cell passage, representing the end of the entire culture sequence, was specifically designated as the "terminal cell." These steps ensured the continuity and standardization of the cell culture process and facilitated accurate analysis of the properties of cells at specific passages. After passaged to the target passage, the cells were cultured as prescribed. Sampling was performed at the next passage, and the cell suspension was used for cell counting, STR typing, target gene sequencing, and target gene copy number detection. Simultaneously, the cell suspension was centrifuged at 500g for 5 minutes, and the supernatant was collected for virus particle count, virus transduction titer, and virus activity detection. The results are shown in Table 2. Experimental results showed that the HEK293-RD114-GP-P037 TCR-producing cell line, which produces γ-retrovirus, could be stably passaged for 8 to 19 generations. The cell viability of HEK293-P037clone46 cells at different passages was within acceptable limits. (See attached figures). Figure 20 The viable cell density of HEK293-P037clone46 cells at different passages was within acceptable limits, as shown in the following figures. Figure 21 The copy number of the target gene in HEK293-P037clone46 cells remained largely consistent across different passages (see results). Figure 22 The analysis of virus particle count results in HEK293-P037clone46 cells at different passages is shown in the figure. Figure 23 The viral transduction titer and viral activity of HEK293-P037clone46 cells at different passages were basically consistent (see results). Figure 24 and Figure 25 ).

[0165] Table 2. Passage stability results of HEK293-RD114-GP-P037 TCR toxin-producing cell line.

[0166]

[0167] Note: *STR typing was commissioned to Suzhou Jianda Biotechnology Co., Ltd.

[0168] #The target gene sequencing was commissioned to Suzhou Genewise Biotechnology Co., Ltd.

[0169] Sequence of this article:

[0170]

[0171] SEQ ID NO: 2 - RD114 protein sequence

[0172] MKLPTGMVILCSLIIVRAGFDDPRKAIALVQKQHGKPCECSGGQVSEAPPNSIQQVTCPGKTAYLMT

[0173] NQKWKCRVTPKNLTPSGGELQNCPCNTFQDSMHSSCYTEYRQCRANNKTYYTATLLKIRSGSLNEV

[0174] QILQNPNQLLQSPCRGSINQPVCWSATAPIHISDGGGPLDTKRVWTVQKRLEQIHKAMHPELQYHPL

[0175] ALPKVRDDLSLDARTFDILNTTFRLLQMSNFSLAQDCWLCLKLGTPTPLAIPTPSLTYSLADSLANAS

[0176] CQIIPPLLVQPMQFSNSSCLSSPFINDTEQIDLGAVTFTNCTSVANVSSPLCALNGSVFLCGNNMAYTY

[0177] LPQNWTGLCVQASLLPDIDIIPGDEPVPIPAIDHYIHRPKRAVQFIPLLAGLGITAAFTTGATGLGVSVT

[0178] QYTKLSHQLISDVQVLSGTIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCFYANKS

[0179] GIVRNKIRTLQEELQKRRESLASNPLWTGLQGFLPYLLPLLGPLLTLLLILTIGPCVFSRLMAFINDRL

[0180] NVVHAMVLAQQYQALKAEEEAQD

[0181] SEQ ID NO: 3 Gag - Pol insertion sequence

[0182] gcggccgcataacttcgtatagcatacattatacgaagttatctgacctcttctcttcctcccacagggcctcgagagatctggcagcggagagggcagaggaa

[0183] gtcttctaacatgcggtgacgtggaggagaatcccggccctaggatgaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggc

[0184] cgtacgcaccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaact

[0185] cttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgg

[0186] gggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccgg

[0187] cccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctgggcagcgccgtcgtgctccccggagtggaggcg

[0188] gccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgagg

[0189] tgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcctgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttcctt

[0190] gaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggca

[0191] ggacagcaagggggaggattgggaagacaatagcactcgacactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgc

[0192] gttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggac

[0193] tttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggta

[0194] aatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggc

[0195] agtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggact

[0196] ttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatc

[0197] gcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctccgcggccccaagcttgggcatcgttctgtgttgtctctgtc

[0198] tgactgtgtttctgtatttgtctgaaaatatgggccagactgttaccactcccttaagtttgaccttaggtcactggaaagatgtcgagcggatcgctcacaaccagt

[0199] cggtagatgtcaagaagagacgttgggttaccttctgctctgcagaatggccaacctttaacgtcggatggccgcgagacggcacctttaaccgagacctcatc

[0200] acccaggttaagatcaaggtcttttcacctggcccgcatggacacccagaccaggtcccctacatcgtgacctgggaagccttggcttttgacccccctccctg

[0201] ggtcaagccctttgtacaccctaagcctccgcctcctcttcctccatccgccccgtctctcccccttgaacctcctcgttcgaccccgcctcgatcctccctttatcc

[0202] agccctcactccttctctaggcgccaaacctaaacctcaagttctttctgacagtggggggccgctcatcgacctacttacagaagaccccccgccttataggg

[0203] acccaagaccacccccttccgacagggacggaaatggtggagaagcgacccctgcgggagaggcaccggacccctccccaatggcatctcgcctacgtg

[0204] ggagacgggagccccctgtggccgactccactacctcgcaggcattccccctccgcgcaggaggaaacggacagcttcaatactggccgttctcctcttctg

[0205] acctttacaactggaaaaataataacccttctttttctgaagatccaggtaaactgacagctctgatcgagtctgtcctcatcacccatcagcccacctgggacga

[0206] ctgtcagcagctgttggggactctgctgaccggagaagaaaaacaacgggtgctcttagaggctagaaaggcggtgcggggcgatgatgggcgccccact

[0207] caactgcccaatgaagtcgatgccgcttttcccctcgagcgcccagactgggattacaccacccaggcaggtaggaaccacctagtccactatcgccagttgc

[0208] tcctagcgggtctccaaaacgcgggcagaagccccaccaatttggccaaggtaaaaggaataacacaagggcccaatgagtctccctcggccttcctagag

[0209] agacttaaggaagcctatcgcaggtacactccttatgaccctgaggacccagggcaagaaactaatgtgtctatgtctttcatttggcagtctgccccagacattg

[0210] ggagaaagttagagaggttagaagatttaaaaaacaagacgcttggagatttggttagagaggcagaaaagatctttaataaacgagaaaccccggaagaaa

[0211] gagaggaacgtatcaggagagaaacagaggaaaaagaagaacgccgtaggacagaggatgagcagaaagagaaagaaagagatcgtaggagacatag

[0212] agagatgagcaagctattggccactgtcgttagtggacagaaacaggatagacagggaggagaacgaaggaggtcccaactcgatcgcgaccagtgtgcc

[0213] tactgcaaagaaaaggggcactgggctaaagattgtcccaagaaaccacgaggacctcggggaccaagaccccagacctccctcctgaccctagatgacta

[0214] gggaggtcagggtcaggagcccccccctgaacccaggataaccctcaaagtcggggggcaacccgtcaccttcctggtagatactggggcccaacactcc

[0215] gtgctgacccaaaatcctggacccctaagtgataagtctgcctgggtccaaggggctactggaggaaagcggtatcgctggaccacggatcgcaaagtacat

[0216] ctagctaccggtaaggtcacccactctttcctccatgtaccagactgtccctatcctctgttaggaagagatttgctgactaaactaaaagcccaaatccactttga

[0217] gggatcaggagctcaggttatgggaccaatggggcagcccctgcaagtgttgaccctaaatatagaagatgagtatcggctacatgagacctcaaaagagcc

[0218] agatgtttctctagggtccacatggctgtctgattttcctcaggcctgggcggaaaccgggggcatgggactggcagttcgccaagctcctctgatcatacctct

[0219] gaaagcaacctctacccccgtgtccataaaacaataccccatgtcacaagaagccagactggggatcaagccccacatacagagactgttggaccagggaa

[0220] tactggtaccctgccagtccccctggaacacgcccctgctacccgttaagaaaccagggactaatgattataggcctgtccaggatctgagagaagtcaacaa

[0221] gcgggtggaagacatccaccccaccgtgcccaacccttacaacctcttgagcgggctcccaccgtcccaccagtggtacactgtgcttgatttaaaggatgcc

[0222] tttttctgcctgagactccaccccaccagtcagcctctcttcgcctttgagtggagagatccagagatgggaatctcaggacaattgacctggaccagactccca

[0223] cagggtttcaaaaacagtcccaccctgtttgatgaggcactgcacagagacctagcagacttccggatccagcacccagacttgatcctgctacagtacgtgg

[0224] atgacttactgctggccgccacttctgagctagactgccaacaaggtactcgggccctgttacaaaccctagggaacctcgggtatcgggcctcggccaagaa

[0225] agcccaaatttgccagaaacaggtcaagtatctggggtatcttctaaaagagggtcagagatggctgactgaggccagaaaagagactgtgatggggcagcc

[0226] tactccgaagacccctcgacaactaagggagttcctagggacggcaggcttctgtcgcctctggatccctgggtttgcagaaatggcagcccccttgtaccctc

[0227] tcaccaaaacggggactctgtttaattggggcccagaccaacaaaaggcctatcaagaaatcaagcaagctcttctaactgccccagccctggggttgccaga

[0228] tttgactaagccctttgaactctttgtcgacgagaagcagggctacgccaaaggtgtcctaacgcaaaaactgggaccttggcgtcggccggtggcctacctgt

[0229] ccaaaaagctagacccagtagcagctgggtggcccccttgcctacggatggtagcagccattgccgtactgacaaaggatgcaggcaagctaaccatggga

[0230] cagccactagtcattctggccccccatgcagtagaggcactagtcaaacaaccccccgaccgctggctttccaacgcccggatgactcactatcaggccttgc

[0231] ttttggacacggaccgggtccagttcggaccggtggtagccctgaacccggctacgctgctcccactgcctgaggaagggctgcaacacaactgccttgatat

[0232] cctggccgaagcccacggaacccgacccgacctaacggaccagccgctcccagacgccgaccacacctggtacacggatggaagcagtctcttacaaga

[0233] gggacagcgtaaggcgggagctgcggtgaccaccgagaccgaggtaatctgggctaaagccctgccagccgggacatccgctcagcgggctgaactgat

[0234] agcactcacccaggccctaaagatggcagaaggtaagaagctaaatgtttatactgatagccgttatgcttttgctactgcccatatccatggagaaatatacag

[0235] aaggcgtgggttgctcacatcagaaggcaaagagatcaaaaataaagacgagatcttggccctactaaaagccctctttctgcccaaaagacttagcataatcc

[0236] attgtccaggacatcaaaagggacacagcgccgaggctagaggcaaccggatggctgaccaagcggcccgaaaggcagccatcacagagactccagac

[0237] acctctaccctcctcatagaaaattcatcaccctacacctcagaacattttcattacacagtgactgatataaaggacctaaccaagttgggggccatttatgataa

[0238] aacaaagaagtattgggtctaccaaggaaaacctgtgatgcctgaccagtttacttttgaattattagactttcttcatcagctgactcacctcagcttctcaaaaatg

[0239] aaggctctcctagagagaagccacagtccctactacatgctgaaccgggatcgaacactcaaaaatatcactgagacctgcaaagcttgtgcacaagtcaacg

[0240] ccagcaagtctgccgttaaacagggaactagggtccgcgggcatcggcccggcactcattgggagatcgatttcaccgagataaagcccggattgtatggct

[0241] ataaatatcttctagtttttatagataccttttctggctggatagaagccttcccaaccaagaaagaaaccgccaaggtcgtaaccaagaagctactagaggagat

[0242] cttccccaggttcggcatgcctcaggtattgggaactgacaatgggcctgccttcgtctccaaggtgagtcagacagtggccgatctgttggggattgattgga

[0243] aattacattgtgcatacagaccccaaagctcaggccaggtagaaagaatgaatagaaccatcaaggagactttaactaaattaacgcttgcaactggctctaga

[0244] gactgggtgctcctactccccttagccctgtaccgagcccgcaacacgccgggcccccatggcctcaccccatatgagatcttatatggggcacccccgccc

[0245] cttgtaaacttccctgaccctgacatgacaagagttactaacagcccctctctccaagctcacttacaggctctctacttagtccagcacgaagtctggagacctc

[0246] tggcggcagcctaccaagaacaactggaccgaccggtggtacctcacccttaccgagtcggcgacacagtgtgggtccgccgacaccagactaagaacct

[0247] agaacctcgctggaaaggaccttacacagtcctgctgaccacccccaccgccctcaaagtagacggcatcgcagcttggatacacgccgcccacgtgaagg

[0248] ctgccgaccccgggggtggaccatcctctagactgacatggcgcgttcaacgctctcaaaaccccttaaaaataaggttaacccgcgaggccccctaatccc

[0249] cttaattcttctgatgctcagaggggtcagtatcgaattcgcggccgct

[0250] SEQ ID NO:4 Gag-Pol protein sequence

[0251] MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWPTFNVGWPRDGTFNRDLITQVK

[0252] IKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVHPKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALT

[0253] PSLGAKPKPQVLSDSGGPLIDLLTEDPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRRE

[0254] PPVADSTTSQAFPLRAGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDC

[0255] QQLLGTLLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYRQ

[0256] LLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETNVSMSFIWQSA

[0257] PDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEEKEERRRTEDEQKEKERDRRR

[0258]

[0259]

[0260] SEQ ID NO 6. 037 TCR α chain

[0261] MASAPISMLAMLFTLSGLRAQSVAQPEDQVNVAEGNPLTVKCTYSVSGNPYLFWYVQYPNRGLQF

[0262] LLKYITGDNLVKGSYGFEAEFNKSQTSFHLKKPSALVSDSALYFCAVRDIEGAGNNRKLIWGLGTSL

[0263] AVNPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAI

[0264] AWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLM

[0265] TLRLWSS

[0266] SEQ ID NO 7. 037 TCR β chain

[0267] MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLV

[0268] SFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSEGQYSYEQYFGPGTRLTVTED

[0269] LRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNY

[0270] SYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS。

Claims

1. Use of cells expressing RD114, Gag, and Pol, or viruses prepared or rescued from said cells, in the preparation of pharmaceuticals. Preferably, the drug is an immunotherapy drug. More preferably, the drug contains immune cells.

2. The use as described in claim 1, characterized in that, The use includes one or more features selected from the following: The cell contains nucleic acid sequences encoding RD114, Gag, and / or Pol; preferably, the nucleic acid sequences encoding RD114, Gag, and / or Pol are integrated into the cell genome. The cell contains an expression cassette containing a nucleic acid sequence encoding RD114 that is operatively linked to a promoter; The cell contains an expression cassette containing nucleic acid sequences encoding Gag and / or Pol that are operatively linked to a promoter. The amino acid sequence of RD114 is shown in SEQ ID NO:

2. The nucleic acid sequence encoding RD114 is operatively linked to the SFFV promoter. The amino acid sequence of Gag-Pol is shown in SEQ ID NO:

4. Nucleic acid sequences encoding Gag and / or Pol are integrated into the AAVS1 site of the cell genome. Nucleic acid sequences encoding Gag and / or Pol are operatively linked to the CMV promoter. The virus is a retrovirus, preferably a gamma retrovirus. The cells in question are HEK293 cells. The cells and / or viruses contain sequences of interest; preferably, the sequences of interest are coding sequences for CAR and / or TCR.

3. A cell with accession number CCTCC NO:C202517.

4. A cell for producing a virus containing a sequence of interest, said cell expressing RD114, Gag, and Pol, said sequence of interest being integrated into the cell genome. Preferably, the cell is a cell with accession number CCTCC NO: C202517 whose genome integrates the sequence of interest. More preferably, the sequence of interest is operatively connected to one or more elements selected from: 5'LTR, splice donor, splice acceptor, and 3'LTR; More preferably, the sequence of interest is a CAR or TCR coding sequence.

5. The cell as described in claim 4, characterized in that, The cell contains one or more features selected from the following: The cell contains nucleic acid sequences encoding RD114, Gag, and / or Pol; preferably, the nucleic acid sequences encoding RD114, Gag, and / or Pol are integrated into the cell genome. The cell contains an expression cassette containing a nucleic acid sequence encoding RD114 that is operatively linked to a promoter; The cell contains an expression cassette containing nucleic acid sequences encoding Gag and / or Pol that are operatively linked to a promoter. The amino acid sequence of RD114 is shown in SEQ ID NO:

2. The nucleic acid sequence encoding RD114 is operatively linked to the SFFV promoter. The amino acid sequence of Gag-Pol is shown in SEQ ID NO:

4. Nucleic acid sequences encoding Gag and / or Pol are integrated into the AAVS1 site of the cell genome. Nucleic acid sequences encoding Gag and / or Pol are operatively linked to the CMV promoter. The virus is a retrovirus, preferably a gamma retrovirus. The cells in question are HEK293 cells.

6. The cell as described in claim 4 or 5, characterized in that, The cells in question are those with accession number CCTCC NO:C202518.

7. A method for preparing the cells of claim 3, the method comprising: A nucleic acid construct containing nucleic acid sequences encoding RD114, Gag, and / or Pol is introduced into cells, the nucleic acid sequences being operatively linked to a promoter.

8. A method for preparing cells according to any one of claims 4-6, the method comprising: (1) An expression cassette containing a nucleic acid sequence encoding RD114, Gag, and / or Pol is introduced into cells, wherein the nucleic acid sequence is operatively linked to a promoter, and the cells are those with accession number CCTCC NO:C202517. (2) Introduce the sequence of interest into the cell obtained by (1); preferably, introduce the sequence of interest into the cell genome obtained by (1). (3) Screening cells, the cells being: (a) The genome contains sequences of interest. (b) It can express proteins encoded by sequences of interest. (c) The virus produced by the cell contains a sequence of interest, or (d) After a virus produced by said cell infects another cell, said other cell is able to express a protein encoded by a sequence of interest. Preferably, step (2) includes the step of introducing the sequence of interest into the cell genome using any of the following technologies: retrovirus, lentivirus, transposon, zinc finger nuclease (ZFN) technology, transcription activator effector nuclease (TALEN) technology, and CRISPR / Cas system.

9. A method for preparing or rescuing a virus, comprising preparing or rescuing the virus using the cells of any one of claims 3-6; Preferably, the method includes: (1) The sequence of interest is introduced into the cell, preferably integrated into the cell genome, and (2) the cell is incubated under conditions suitable for virus production. More preferably, step (1) includes the step of introducing the sequence of interest into the cell genome using any of the following technologies: retroviruses, transposons, zinc finger nucleases (ZFNs), transcription activator effector nucleases (TALENs), and the CRISPR / Cas system. More preferably, the virus is a retrovirus, and / or the cell is a HEK293 cell.

10. Use of the cell according to any one of claims 3-6 in the preparation of viruses, increasing virus titers, and improving virus packaging efficiency.

11. A kit comprising the cells of any one of claims 3-6.

12. A pharmaceutical composition comprising the virus prepared by the method of claim 9 and pharmaceutically acceptable excipients.

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